Journal of Drug Targeting

ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20

Liposome: composition, characterization,

preparation, and recent innovation in clinical
applications

Kamel S. Ahmed, Saied A. Hussein, Abdelmoneim H. Ali, Sameh A. Korma,

Qiu Lipeng & Chen Jinghua

To cite this article: Kamel S. Ahmed, Saied A. Hussein, Abdelmoneim H. Ali, Sameh
A. Korma, Qiu Lipeng & Chen Jinghua (2018): Liposome: composition, characterization,
preparation, and recent innovation in clinical applications, Journal of Drug Targeting, DOI:
10.1080/1061186X.2018.1527337

To link to this article: https://doi.org/10.1080/1061186X.2018.1527337

Accepted author version posted online: 21

Sep 2018.

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Liposome: composition, characterization, preparation, and recent innovation in clinical

applications

Kamel S. Ahmed a,b, Saied A. Hussein c, Abdelmoneim H. Ali d, Sameh A. Korma d, Lipeng Qiu a*,

Jinghua Chena*

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a
Department of Pharmaceutics, School of Pharmaceutical Sciences, Jiangnan University, 1800 Lihu

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Road, Wuxi 214122, Jiangsu, PR China

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b
Department of Pharmaceutics, Faculty of Pharmacy, Minia University, Minia 11432, Egypt
c an
Biomaterial Department, College of Life Science and Technology, Huazhong University of

Science and Technology, Wuhan 430074, Hubei, PR China

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d
State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food

Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu
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Road, Wuxi 214122, Jiangsu, PR China

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* Corresponding authors:

Lipeng Qiu Email: flyqlp@163.com

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Tel: +86-17701518734 fax: +86-17701518734

Jinghua Chen Email: chenjinghua@jiangnan.edu.cn

Tel: + 86-51085911900 fax: +86-51085329042

Abstract

In the last decades, pharmaceutical interested researches aimed to develop novel and

innovative drug delivery techniques in the medical and pharmaceutical fields. Recently,

phospholipid vesicles (Liposomes) are the most known versatile assemblies in the drug delivery

systems. The discovery of liposomes arises from self-forming enclosed phospholipid bilayer upon

coming in contact with the aqueous solution. Liposomes are uni or multilamellar vesicles consisted

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of phospholipids produced naturally or synthetically, which are readily non-toxic, biodegradable,

and are readily produced on a large scale. Various phospholipids, for instance, soybean, egg yolk,

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synthetic, and hydrogenated phosphatidylcholine consider the most popular types used in different

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kinds of formulations. This review summarizes liposomes composition, characterization, methods
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of preparation, and their applications in different medical fields including cancer therapy, vaccine,

ocular delivery, wound healing, and some dermatological applications.

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Keywords: Liposomes; phospholipids; applications; drug delivery; cancer; wound healing.

Abbreviations:
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PC: phosphatidylcholine; PE: phosphatidylethanolamine; PS: phosphatidylserine; PI:

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phosphatidylinositol; PA: phosphatidic acid; PG: phosphatidylglycerol; SMs: Sphingomyelins;

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LPC: lysophosphatidylcholine; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP:

1,2-Dioleoyl-3-trimethylammonium propane; MPER: membrane-proximal external region; DOPC:

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1,2-Dioleoyl-sn-glycero-3-phosphocholine; DOPG: 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-

glycero1; DPC: dimethyl dioctadecyl ammonium, Poly I: C and cholesterol; bFGF: basic fibroblast

growth factor; TRT: tretinoin; LSSPL: liposomes loaded with lipase-sensitive singlet oxygen-

producible (pullulan-pheophorbide a (PU-Pheo A); PUVA: Psoralen combined with ultraviolet A

radiation.
1 Introduction

Liposomes are one of the main advanced approaches in drug delivery systems. Researches

related to liposomes have gained an attractive importance in pharmaceutical, biological, and

medical fields since liposomes consider the most proper carriers for the introduction of all kinds of

agents, such as anticancer drugs [1, 2], antibiotics [3, 4], anti-inflammatory [5, 6], genes [7, 8], and

antifungal [9, 10]. Liposomes are the first enclosed microscopic phospholipid bilayer systems,

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which were approved in 1965 [11, 12]. They are circular soft-matter vesicles formed from one or

more bilayer membrane(s) separating an aqueous medium from another. Phospholipid molecules

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mainly composed of different polar head groups and two hydrophobic hydrocarbon chains. The

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polar groups can be zwitter ionic or negatively charged. The hydrocarbon chain molecules have
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different lengths and possess different degrees of unsaturation. The formation of liposomes occurs

spontaneously upon reconstitution of dry lipid films in an aqueous solution [13, 14].
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The main advantages of systemic liposomes as drug formulations arise from their

biodegradability, lowers systemic toxicity, targeted delivery, sensitive molecules protection, and
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improved pharmacokinetic effects. On the other hand, their advantages for the topical applications
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accrue from the demonstrated ability to decrease serious incompatibilities and side effects that
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might originate from the undesirably large systemic drugs absorption, the significant enhancement

of drug accumulation at the required site of action due to the high similarity between the liposome
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composition and the biological membranes [15].

Many types of research have been carried out for the surface modification of liposomes to

enhance their properties and increase their applicability to deliver the active pharmaceutical

ingredients to the site of action for in-vivo applications. For example, the utilization of polyethylene

glycol (PEG) and polyvinyl alcohol (PVA) decreased opsonization and made the liposomes less
susceptible for hepatic clearance, and consequently prolonged liposomes residence time in the

systemic circulation [6, 16]. As well, the insertion of rhodamine-123-conjugated polymer in the

surface of the liposomes increased the formulation accumulation in the mitochondria [17]. The

incorporation of D-α-tocopheryl polyethylene glycol 1000 succinate-triphenylphosphine conjugate

(TPGS1000-TPP) to the surface of a liposomal formulation of paclitaxel improved its cellular

uptake and mitochondrial targeting and consequently enhanced apoptosis processes in drug-

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resistant lung cancer [18]. The previously mentioned advantages and the simple handling and

modulation processes of liposomes explain their strong invasion for the scientific research area and

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various biological applications and also explain the abundant presence of liposome preparations in

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market and clinical trials. there are a number of liposomal products such as ambisome, myocet,
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doxil, depoCyt, etc., approved by the FDA for commercial usage. Table 1 showing some liposomal

formulation recently used in the global liposomes drug delivery market and some others still under
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clinical trials.

2 Composition of liposomes
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Liposomes are mainly composed of phospholipids, which contain two major categories
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including glycerophospholipids and sphingomyelins. The phospholipids of eukaryotic cells are

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mainly glycerophospholipids, in which glycerol considered the backbone. The chemical structure of

glycerophospholipids consisted of a hydrophilic head group and a hydrophobic side chain. Different
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glycerophospholipids are obtained as a result of head group variation, for example,

phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),

phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin (CL)

[19, 20]. As well, the length variation of the nonpolar moieties results in various
glycerophospholipids, such as dimyristoyl, dipalmitoyl, or distearoyl PC. Furthermore, the bonding

type (ether or ester) between glycerol and aliphatic chains results in different glycerophospholipids.

Sphingomyelins (SMs) are a vital membrane component of the animal cells [21, 22].

Sphingosine is the backbone of SM; each molecule of SM averagely has cis-double bonds in amide-

linked acyl chains. The natural SMs have typical acyl lengths usually longer than the paraffin

residues of sphingosine, so they considered as asymmetric molecules. SMs have the capability to

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form intermolecular and intramolecular hydrogen bonds. SMs have been used in the liposomal

formulation and showed good entrapment efficiency, greater serum stability, with a rapid release

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profile in comparison with the DSPC liposomes [23]. Another study showed that a spherically

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shaped vesicle with small particle size has been successfully prepared from sphingomyelin solution
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by using ultrasonic–supercritical CO2 technique but the size distribution of the vesicles increased
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by increasing the operating temperature as a result of aggregation [24]

2.1 The abundant sources of phospholipids

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Animal tissues (bovine brain and egg yolk), and vegetable oils (soybean, corn, cotton seed,
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sunflower, and rapeseed) are considered the common phospholipids sources. Soybean oil and egg
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yolk are the common important sources of phospholipids. Egg yolk is distinguished with (1) larger
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quantities of PC, (2) phospholipids with long-chain polyunsaturated fatty acids (arachidonic acid
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and docosahexaenoic acid), (3) the presence of SM, (4) egg yolk lecithins of the higher saturation

level, which could cause better have been used for phospholipids extraction [25]. In a recent study,

ethanol and butanol with different contents of water were assessed for phospholipids extraction.

The yolk flakes were prepared by using a small drum dryer heated with saturated steam. The liquid

yolk was applied on the smooth hot surfaces of the drums, where denaturation of yolk protein and

water evaporation takes place. The dried yolk was collected as “flakes” from the surface of the
drum by a scraper, and the dried flakes were then packed into a column through which the organic

solvent from the reservoir was added to submerge the flakes, followed by solvent evaporation from

the lipid extract under vacuum by using a rotary evaporator. Then, the lipid extracts were dried at

40 °C for 5 h in a vacuum oven. All yolk lipids were effectively extracted by using butanol with a

little preference for phospholipids, while lipids extraction by using aqueous ethanol was affected by

its water content since ethanol of 75% exhibited the highest preference for phospholipids [26]. In

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another study, three solvents (butanol 100%, butanol 80%, and ethanol 95%) were used for the

extraction of phospholipids by injecting the yolk as a thin stream into the hot solvent, after that the

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liquid yolk was solidified upon contact with the solvent into the thread. 100 g of egg yolk was

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vortexed and texturized in 200 mL of the solvent under similar conditions. The de-oiled yolk thread
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was subjected to extraction 4 more times sequentially, then the solvent was evaporated using a

rotary evaporator, and the obtained lipids were dried at 40 °C for 5 h in a vacuum oven. It was
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shown that the total yolk lipids were effectively extracted by using butanol, and the extraction was

faster than that of ethanol, but with little preference for phospholipids. Conversely, ethanol had a
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more preference for phospholipids than for neutral lipids [27].

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Liu, Zhou [28], extracted phospholipids from edible clams (Cycling Sinensis, Mactra
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Chinensis Philippi, Mactra veneriformis Reeve, Meretrix meretrix, Saxidomus purpurata, and

Ruditapes phliippinarum) by using a mixture of hexane and ethanol. Clam meat powder was mixed
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with hexane/ethanol (1:1, v/v), and vortexed for 1 min, stirred at 50 °C for 90 min, and then the

mixture was centrifuged at 7,800 g for 10 min. Finally, the organic layer was collected and dried at

35 °C under nitrogen. Large quantities of the polyunsaturated fatty acids (docosahexaenoic acid and

eicosapentaenoic acid) were recovered. The extracted oils contained a high percentage of
phospholipids making up 39.86–74.05% of the total lipids. PC (37.40–52.19%) and PE (34.74–

43.10%) were the predominant species.

Phospholipids classes from duck, quail, and hen egg yolks were separated and identified.

Egg yolk was separated from the egg white and transferred into a 200-mL vial, followed by the

addition of ethanol, and stirring for 10 min with a magnetic stirrer. The solvent layer was filtered

into a round-bottom flask, and the residual was extracted with ethanol twice. The ethanolic solution

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was crystallized under the lower temperature at 2–5 °C for 2 h, then ethanol was evaporated from

the solution by a rotary evaporator, and the extracts were collected. The residue was then dissolved

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in hexane and transferred into a vial placed in an ice bath. Finally, 120 mL of cold acetone (-20 °C)

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was carefully poured into the stirring mixture until phospholipids precipitation. The obtained results
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revealed that 57 molecular species of egg yolk phospholipids were detected and identified. Among

the different classes of egg yolk phospholipids, PC (16:0–18:1), PE (18:0–20:4), PI (18:0–18:2), PS

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(18:0–18:2), SM (d18:1/16:0), and lysophosphatidylcholine (LPC) (16:0) were the predominant

molecular species [29].

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Gas chromatography coupled with mass spectrometry (MS) and hydrophilic interaction
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liquid chromatography was used for the determination of omega fatty acids profile in egg yolk.
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Separation was accomplished by the liquid chromatography by using columns (stainless steel)

packed with a homemade alkyl-amide stationary bonded phase (synthesized on Kromasil 100 silica
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gel). A diode array detector (SPD-M20A) was used for measuring the absorbance at 206 nm.

MS/MS analysis was achieved under the positive ionization mode on a triple quadruple coupled

with an electrospray ionization source. During 20 min the phospholipids were eluted in the

following order: PG, LPG, PE, LPE, PC, SM, and LPC. Furthermore, palmitic acid was the major

saturated fatty acid detected [30].

 Phospholipids fractions were extracted from Camelina sativa seeds by using the

supercritical carbon dioxide method. A total phospholipid content of 360 and 130 mg/kg lipid was

obtained by the hexane-extracted and cold-pressed oils, respectively. While the total content of the

phospholipid extracted with pure supercritical carbon dioxide method ranged between 520 (45

MPa/70 °C) and 2000 mg/kg lipid (45 MPa/50 °C). PI, PE, and PC were the major classes of

phospholipids found in the lipids of camelina seeds, and PI was the predominant species. More

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phospholipids were extracted by increasing the concentration of the ethanol to 10% (w/w) [31].

3 Methods of liposomes preparation

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All the methods of liposomes preparation involve three to four basic steps:

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1. Drying lipids through the evaporation of the organic solvent.

2. The lipid dispersion in an aqueous media.

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3. Liposome purification.
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4. Analysis of the final product.

The preparation of liposomes could be achieved by using three different techniques, such as the
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mechanical methods, the solvent dispersion methods, and methods based on fusion or size
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transformation of the prepared vesicle [32].

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3.1 Mechanical methods

Preparation of liposomes by the thin film hydration method

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The most common simply and widely applicable method for liposomes formulation is the

thin-film hydration method [33]. In this technique, liposomes are prepared by dissolving the lipid in

an organic solvent usually chloroform or mixtures of chloroform and methanol, and the solvent is

then removed by film deposition under vacuum. After the complete evaporation of the organic

solvents, the lipid residue is hydrated by using an aqueous buffer; the lipids spontaneously undergo
swelling and hydration to form a liposome. This method produces liposomes with a heterogeneous

sized aggregation of multilamellar vesicles over one micrometer in diameter. The particles can be

downsized by using different techniques, for instance, extrusion or sonication [34, 35].

3.2 Preparation of liposomes by solvent dispersion methods

3.2.1 Ether injection method

In this method, liposomes are prepared by dissolving the lipid in diethyl ether or a mixture of

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methanol and ether, followed by the slow injection to an aqueous solution of the compound to be

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incorporated under lower pressure or at 55-65 °C. The main drawbacks of this procedure are the

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heterogeneous population of the obtained vesicles, and the exposure of the substances to be

encapsulated to high temperature [36].

3.2.2 Ethanol injection method

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This method involves the utilization of ethanol to dissolve phospholipids and cholesterol. By

means of a syringe pump, the resulting lipid solution is injected under stirring conditions in a
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definite volume of the aqueous solution. Liposomes are then spontaneously formed as soon as the
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lipid solution contacts the aqueous phase. The suspension of liposomes is then left under stirring at

room temperature for 15 min. This procedure offers numerous advantages, such as fast
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implementation, simplicity, and reproducibility. Also, it does not lead to oxidative alterations or
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degradation of lipids and has the ability to form small unilamellar vesicles without extrusion or

sonication [37, 38].

3.3 Preparation of liposomes by methods based on fusion or size transformation of prepared

the vesicle

3.3.1 Freeze-thaw extrusion method

Freezing and thawing method is considered to be a convenient technique for increasing the

trapped capacity of the liposomal preparations since it reveals a physical disruption of the lamellar

structure, likely due to the ice crystals formed during the process of freezing [39]. Liposomes

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prepared by the film method were vortexed with the material to be incorporated until the whole

lipid film is suspended, and the resulted vesicles are frozen in warm water and vortexed again. Then

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extrusion of the sample is applied three times after 2 cycles of freeze-thaw and vortexing, followed

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by 6 cycles of freeze-thaw and additional 8 extrusions. Freeze-thaw cycling is a method frequently
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used in liposomes preparation in order to increase the encapsulation efficiency [40, 41].

3.3.2 The dehydration/rehydration method

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One of the drawbacks of macrolide and aminoglycoside antibiotics liposomes is their low
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encapsulation efficiency, which results in preparations with lower drug content. The encapsulation
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efficiencies and stability (in-vitro) of these antibiotics could be enhanced by

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dehydration/rehydration of the obtained vesicles [42]. Emptying the buffer containing small
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unilamellar vesicles and rehydrating it with the aqueous solution containing the compound to be

incorporated after which they are dried. This lead to solid lipids dispersion in small subdivided
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forms and the vesicles are then rehydrated. Liposomes prepared by this technique are usually

oligolamellar vesicle [43, 44].

3.4 Supercritical fluid technology

The supercritical state of a fluid is intermediate between that of gas and liquids. In the

pharmaceutical field, supercritical carbon dioxide (scCO2) is the most commonly used gas which

can become supercritical at its critical temperature and pressure of 31.1°C, 7.38 respectively [45].

The supercritical anti-solvent (SAS) method.

This technique is being used to produce a micronized and homogeneous dispersion of phospholipid

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materials. Cholesterol and other lipid materials dissolved in an organic solvent and placed in a glass

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container. CO2 gas is sprayed through capillary tubes into a high-pressure precipitation vessel and

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as a result of a sudden change in temperature and pressure the CO2 gas transformed into a

supercritical phase. Subsequently, evaporation of the organic solvent takes place and the lipids are
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extracted into the supercritical phase, which leads to supersaturation of the lipids in the

scCO2 phase and precipitation of the lipid materials. After that, the organic solvent is removed
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by continuous pumping CO2 into the vessel to achieve fine lipid particles. Finally, adding the
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aqueous solution for liposomes formation. A previous study made a comparison between the
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conventional method (Bangham method) and the supercritical anti-solvent (SAS) method, it was
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reported that SAS method was more efficient and environmentally-friendly procedures to produce
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liposomes. Because it produced small particle size distribution and high entrapment efficiency and

considered as an environmentally-friendly technique because it enables to apply “soft” organic

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solvents for example ethanol if compared with the conventional methods organic solvents

(isopropyl ether, chloroform, diethyl ether, and methanol). And also, the SAS process is performed

under lower temperature conditions unlike the conventional Bangham method [46]. Another study

used the Supercritical process for encapsulation of both eugenol (EUG) and α-lipoic acid (ALA) as

an antioxidant in liposome vesicles. It was mentioned that EUG was entrapped with an overall
efficiency of 86.3% with a particle size of 200 nm and ALA was encapsulated with a maximum EE

of 68.1% with a particle size of 230 nm and the prepared liposome exhibited a good stability for at

least 40 days [47]. The previous studies did not show a significant difference in terms of the

liposome size, entrapment efficiency and stability compared with the conventional Bangham

method for liposome preparation except SAS process lead to complete removing of the organic

solvent. To avoids the problems caused by organic solvent residues, and due to the dissolution

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properties of supercritical CO2, that could be used as an excellent substitute for the organic solvent,

an improved Supercritical reverse phase evaporation (ISPER) method was developed in which the

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aqueous solution with the phospholipid materials were introduced into a sealed viewing cell.

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followed by adjustment the temperature and pressure to suitable values and then introducing the
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CO2. After equilibration, CO2 removed, and liposomes were formed, and the liposome prepared by

this method showed improvement in entrapment efficiency of glucose using alpha-dioleoyl

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phosphatidylcholine (DOPC) if compared by the Bangham method. moreover, the liposome

prepared by the ISPER method was highly stable for 30 days [48]. In general, by comparing the
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SCF technique with other conventional methods for liposome preparation the SCF has provided
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several advantages for example, due to the use of supercritical carbon dioxide (scCO2) (non-
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flammable, inert, non-toxic, and more economic), SCF was considered as a green process,

Possibility of large-scale production of liposome by SCF under the good manufacturing practice
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(cGMP) conditions [49], finally liposome once formed no need for further processing (drying and

precipitation) to achieve dry powder liposomal formulations [50].

4 Liposome characterizations

4.1 Particle size

The liposomes size considers one of the vital factors that should be controlled particularly

when these formulations are intended for the parenteral application (topical, injection, and

inhalation). Numerous procedures are available for monitoring the liposomal size, for example,

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Sonication, Extrusion, and Homogenization.

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Sonication

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The multilamellar vesicles (MLVs) suspension that produced by different preparation techniques

(thin film hydration method) is transferred into test tubes and subjected for sonication by tip
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sonication that require larger sample volume (1-5 ml) and more energy or in a bath sonicator that

provide better control of the temperature, small volumes of the sample is required (1 ml), and
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proper for preparation that in jelly form or that doesn't swell well. The pressure stress induces
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disruption of the large and the MLVs present in the samples into small unilamellar vesicles.
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Sonication process normally lasts for 5–10-min and this will provide small unilamellar vesicles
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(20-50 nm). But the different kind of the lipids will impact on the vesicles final size, for example,
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diacyl cationic lipids can form micelles (dioctadecylamidoglycylspermine (DOGS)), and neutral
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lipid (Dioctadecyl diammonium bromide (DOBAD)), the vesicles cannot be downsized <130 nm

[51, 52].

Extrusion

Liposome extrusion occurs by pushing the large MLVs solution through filters of polycarbonate

membrane. The desired vesicles size controlled by the applied pressure, since the average size
decreases upon increasing the extrusion pressure [53], also decreasing the pore size of the filter

membrane result in reduction in size and size distribution of the liposomes, it was reported that

using filter membrane with pore size larger than 0.2 μm the size of the obtained liposomes was

smaller than the membrane pore size, but by using a membrane with a pore size smaller than 0.2

μm, the size of the produced liposomes was slightly larger than the filter pore size [54]. And also,

another study proved that filtering of the MLVs solution using a filter membrane with pore size

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∼1 μm followed by five times repeated filtration through 0.4- and 0.2-μm pores. Then five to ten

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extrusions using a 100-nm membrane pore size will result in the formation of large unilamellar

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vesicles with size ∼110–120 nm. If the size is needed to be smaller, continuous filtration using a

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membrane filter with 80- and 50-nm pores size is required. The vesicles treated by the extrusion
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techniques characterized by proper homogeneity and easy to control the vesicles size distribution,

especially for vesicles of large diameters (100–500 nm) [51].

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Homogenization
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The downsizing of the liposomes by this technique established by the collision of the
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larger vesicles in the interaction chamber at high pressure [55]. This method is characterized by

its simple usage for large-scale production of the liposome, high capacity (10 mL to hundreds of
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liters), and rapid procedures. But the probability of contamination and degradation of the sample
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can occur especially with very small and certain large vesicles. Normally, minimal size can be

attained by 3-5 passages through the interaction chamber, the diameter and the entrapped volume

of the vesicles decreases with increasing the cycles number and the inlet pressure [56]. For

assessing the liposome size and size distribution, numerous methods are available, for example,

size-exclusion chromatography (SEC), field-flow fractionation and static or dynamic light

scattering, microscopy techniques.

As a result of the drawbacks of the electron microscope technique (EM) (TEM, cryo-EM, freeze

fracture TEM) like complicated sample preparation (staining and drying procedures), induce

shape alteration and shrinkage, and time-consuming, so atomic force microscopy (AFM) is a

novel rapid techniques that able to assess the three-dimensional shape of the liposome surface

without sample modification with a sharp probe or tip, and doesn't cause any damaging or

alteration effects on the liposome, but liposome dispersion analysis should be immediately after

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deposition of the sample because evaporation of the aqueous medium will lead to vesicles

rearrangements. Field-flow fractionation (FFF) is a flexible elution method, provides convenient

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and rapid procedures for measurement and separation of liposome size. It has the ability to separate

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a wide range (1 nm – 100 μm) of particle sizes with high resolution. In this technique Particle size
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separation depending on hydrodynamic size basis (flow FFF) or weight basis (sedimentation FFF).

Furthermore, when joined to other detectors like light scattering, transmission electron
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microscopy, UV-absorbance, and atomic force microscopy can offer a proper information on

vesicle properties such as size, structural parameters, shape, and sample contamination [51, 57].
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Size exclusion chromatography (SEC), is a standard technique used for size separation, Separation
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occurs by passing the sample dispersion through a column packed with a porous material that
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entraps small particles and excludes large particles from the internal pore volume resulting in their

lower retention on the column. This result in larger particles firstly eluted before the smaller one. It
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is characterized by the following: simple and uncomplicated technique, small factors number

required to be controlled for the experiments, simple set up, and its coupling with Refractive index,

UV-Vis, or fluorescence detectors makes it able to rapid and easy method validation and

development but there are some drawbacks, it deals with a small molecular weight range of

separation and rigidity of the analysis conditions and high sensitivity of the separation method to
several parameters like ionic strength, pH, and type of charge (cation and anion) of the carrier

solution [58].

Dynamic light scattering

The intensity of light scattered by suspended particles undergoing Brownian motion (from

collisions between suspended particles and solvent molecules) can be determined as a function of

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time by Dynamic light scattering (DLS). This technique provides several advantages such as

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Accurate, repeatable and reliable size analysis in its native environment, high concentration and

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turbid samples can be directly measured, fully automated measurement, simple set up, measurement

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of small sizes < 1nm and MW < 1000Da, and Low sample volume (as little as 2µL) [59, 60, 61].

4.2 Encapsulation efficiency

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Increasing the drug encapsulation efficiency (EE) will contribute to the enhancement of the drug
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bioavailability [62]. Different methods used for measurement of the encapsulation efficiency.
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Firstly separation of free drug from loaded one occurs by different separation techniques
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(centrifugation and dialysis membrane), and then direct determination of encapsulated drug
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achieved by solubilizing or disruption of the lipid bilayer by methanol [63, 64], chloroform [65, 66]
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or by triton x 100 [67, 68] followed by filtration and measurement. Indirect methods achieved by
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determining the quantity of unencapsulated drugs then subtract the value from the total amount of

the drug used [69]. A recent study using liquid chromatography (nanoparticles exclusion

chromatography) for rapid measurement of encapsulation efficiency without any pretreatment for

the sample through direct injection of the liposomal suspension into the column, and the free drug

eluted according to its physical properties under appropriate mobile phase conditions [70, 71].

Another study used different techniques [(solid-phase extraction (SPE), size-exclusion

chromatography (SEC), hollow fiber centrifugal ultrafiltration (HF-CF-UF) and centrifugation

ultrafiltration (CF-UF)] for assessment of the EE of the Amphotericin B (AmB) loaded liposome.

The EE by SPE was found 5–13%, SEC was about 93%, and by HF-CF-UF was approximately

99.0% and nearly 100% by CF-UF. The paper reported that the variation of EE among these

methods back to the following reasons; by using SPE, the interaction of cholesterol with the

stationary phase result in difficult elution with water, and raise the leakage probability of liposome,

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by SEC some parameters could cause liposome leakage, such as osmotic shocks, adsorption, the

pore size and the kind of columns. While for CF-UF, the main drawback prevents the

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unencapsulated drug to pass freely through the membrane, leading to no detection of the

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unencapsulated drug, was the concentration polarization, HF-CF-UF could actually reflect the
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encapsulation efficiency of the liposomes with the unentrapped AmB concentration less than 25.0

micro g/mL, but by increasing the concentration more than 25.0 micro g/mL, lead to an increase in
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the size of AmB molecular aggregates and consequently entrapped by hollow fiber [72]. There are

different substances that have an effect on the encapsulation efficiency, for example, cholesterol,
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several studies proved that the encapsulation efficiency decreased by increasing the cholesterol
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content, and these may result from the steric hindrance and the competition between the cholesterol
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and hydrophobic drug molecules for space present in the phospholipids bilayer resulting in lower

encapsulation of the drug molecules [66, 73]. Another study investigated the effect of aptamer on
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encapsulation efficiency. Aptamers are DNA or RNA sequences produced to display high affinity

and specificity against a broad range of targets with poor immunogenicity, high thermal stability,

and synthesis on large scale with lower costs for production, it was reported that the loading

efficiency of doxorubicin-binding aptamers was ten folds greater than conventional doxorubicin

liposomal formulations and also encapsulation efficiency of tobramycin (hydrophilic) into

liposomes was improved 6 times by applying aptamers [74]. Another study confirmed that the

presence of transmembrane salt gradient (ammonium salts, sodium salts) had a strong effect on the

EE. It was investigated that using of salt gradients means for loading of Doxorubicin result in

charging and protonation of DXR inside the liposome, and also DXR precipitation in the interior

part of the liposome when the DXR concentration exceeded the solubility. Also showed that the use

of ammonium salt-gradients (NH4+-citrate 100%, NH4+-phosphate 98%, NH4+- sulfate 95%,

t
NH4+-acetate 77%) had higher loading efficiency than sodium salt gradient (Na+- citrate 54%, Na+-

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phosphate 52%, Na+- sulfate 44%, Na+- acetate 16%) [75, 76].

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4.3 Liposomal Drug release

Release studies can be performed at 37 oC under sink condition using appropriate release
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media (buffer). a dialysis membrane with specific cut-off molecular weight used in the form of
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dialysis bag or on the end of the tube and soaked in receptor media under stirring condition. The

release medium is usually buffer with PH 7.4 and perfect sink conditions and kept at 37 oC under
d

stirring to mimic the in vivo conditions (In-Vitro Drug Release). At predetermined time intervals,
e
pt

aliquot volumes of the medium were taken out at various time intervals for concentration detection

by different analysis technique (HPLC, UV-vis spectrophotometry), and substituted with the same
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volume of fresh media for keeping the volume of the receptor media constant. The release of the
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drug from liposome is affected by several parameters. For instance, the presence of cholesterol in

liposome formulation will influence on the release of the hydrophilic drugs as the quantity of

cholesterol increases, the release will be rapid. Conversely, the release of the hydrophobic drugs

exhibited a reduced release action [77]. Another study confirmed that the incorporation

of cholesterol into the doxorubicin liposome decreased the undesired leakage of doxorubicin at

37°C in buffered saline and also in fetal bovine serum (FBS) [78]. Moreover, the drug release can
be controlled to overcome the problems that arise from conventional liposome like rapid leakage,

slow release of the entrapped drug, the drug cannot be controlled released in targeting sites, or the

drug may be leakage before reaching the active sites and these drawbacks can be avoided by

incorporating components in liposome formulations to achieve pH [79, 80, 81], thermal [82, 83,

84], photothermal [85, 86], enzymatic [87, 88, 89], or magnetic [90, 91, 92] triggered release.

4.4 Zeta Potential

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The ZP refers to the potential difference between the electric double layer (EDL) (an adsorbed

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double layer developed on the surface of dispersed charged particles) of the movable particles and

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the layer of dispersant around them at the slipping plane. The particle surface charge value reflects

the nanosuspensions stability, considering that NP-dispersions with ZP values of ±30 mV is

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electrostatically stabilized nanosuspension [93]. For measuring the zeta potential, an electric field
M
is applied, and the particles electrophoretic mobility is measured based on the scattering of an

incident laser by the mobile particles. Many factors affecting zeta potential value, for instance, PH,
d

ionic strength, and the particles concentration [94]. The phospholipid composition (positively and
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negatively charged phospholipids) of liposomes is the main content that influences on the overall

surface charge of the liposome [20].

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4.5 Freeze drying

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Liposomal preparations do not meet the required long-term stability standards of

pharmaceutical formulations if stored as aqueous suspensions. Over time the incorporated active

ingredients tend to leak out of the lipid bilayer structure and vesicles liable to fuse or aggregate on

storage. So, the removal of water will improve the stability and increase the shelf life of liposomal

formulations. Freeze drying is one of the most commonly used methods for drying and enhancing
the stability of several pharmaceutical products including liposomes. The drying process includes

three separate steps, begin with freezing then ice sublimation and finally desorption of unfrozen

water, however during these stages physical changes of the vesicles such as alterations in the

vesicle size and loss of the incorporated agent may occur. Fortunately, these obstacles were solved

by using sugars such as sucrose, glucose, trehalose, mannitol, and lactose that have the ability to

interact with the headgroups of the phospholipid through formation of H-bonds leading to

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depression of phase transition temperature (Tm). Consequently, phase transitions during

dehydration and rehydration will be avoided and the loss of incorporated solutions will be

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prohibited. Another mechanism of the lyoprotectant is the formation of viscous glassy matrix in and

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around the liposomes preventing the fusion process and protects vesicles against rupture during
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formation of ice crystals [95]. The influence of different cryoprotectants on the stability of liposome

preparation of 5-Fluorouracil was studied. The data showed that lyophilized cake of liposomes
M
(without cryoprotectants) was compact and hard to reconstitute, on the other hand, when using

cryoprotectants the liposome cake was fluffy and easily reconstituted. Glucose, mannitol and
e d

trehalose were used as cryoprotectants. The protective efficacy of trehalose was more than glucose
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and mannitol, the efficiency of the lyophilization process increased with increasing the
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cryoprotectant concentration [96]. Sucrose was used as cryoprotectant in liposome preparation of

vitamin C and hydrophobic drug medium-chain fatty acids (MCFAs). liposomes were dried below
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−86 °C for 48 h at a sucrose/phospholipids ratio of 2:1 (w/w). The dried liposomes exhibited high

encapsulation efficiency of MCFAs and vitamin c (44.26 ± 3.34, 62.25 ± 3.43 % respectively) with

small particle size (110.4 ± 7.28) nm and high storage stability at 4 °C for 60 days [97]. The

stability properties of liposomal preparation incorporating ftorafur and vitamin A was investigated

in presence of suitable sugars, the transition temperature of liposomal dispersions was the highest
with trehalose and the liposome size was smallest (294±8nm) than that of glucose (751±12nm),

sucrose (426±9nm), and mannitol (356±4nm), moreover, the liposome suspension with 15%

trehalose exhibited the highest retention rate 60.5 and 99.2±0.1% for ftorafur and vitamin A

respectively after freeze-drying [98]. Recently published study reported that trehalose at a 5:1 sugar

to lipid ratio had the ability to maintain the permeability properties and the structural integrity of the

prednisolone sodium phosphate liposome during the freeze-drying procedure [99]. Many factors of

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the formulation should be carefully investigated to ensure the success of freeze-drying process as

the liposome composition (phospholipid type, concentration and type of conjugated polymers, type

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and exact concentration of cryoprotectants, interaction between cryoprotectants and liposomes,

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surface modification of liposomes).

4.6 Stability study

an
Stability of liposome is a complex issue. It can be classified into chemical, physical and
M
biological stabilities which are strongly inter-related to each other, the physical and chemical
d

stability influence on the liposomes-shelf life (size distribution, entrapment efficiency, and
e

compound degradation), Physical stability studies of liposomes can be achieved either by visual
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appearance and observations through determination of color changes and sedimentation of liposome
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formulations or microscopic observations by using transmission electron microscopy and atomic

force microscopy (AFM) to evaluate the particle size and the three-dimensional shape of the
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liposome surface, vesicle surface charges play an important role in preventing aggregation and also

optimizing the storage conditions (temperature and humidity) [100] help in extending the liposome

shelf life stability. the chemical stability of the phospholipids from which the bilayer of the

liposome is composed is very important and should be considered especially if the vesicle

composed from unsaturated phospholipids that prone to hydrolysis and oxidation. The chemical
stability studies can be achieved by studying the phospholipid composition (hydrolysis and

peroxidation) and drug degradation using liquid chromatography, thiobarbituric acid (TBA) test

(lipid oxidation), and spectroscopy. Protection of phospholipids against oxidation can be attained by

using high-quality lipid materials void from hydroperoxides and metal ions. Liposome preparation

under an inert gas such as argon or nitrogen reduces the oxidation of lipids, Storage of liposome at

low temperatures and away from light and oxygen will decrease the chance of oxidation. Moreover,

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using antioxidants, such as butylhydroxytoluene (BHT) and a-tocopherol can protect against

oxidation. phospholipids hydrolysis can completely be avoided by water removal through freeze-

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drying in the presence of a suitable stabilizing cryoprotectant [101, 102, 103]. Stability of liposome

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inside the biological fluids is mainly affected by several blood components including lipoproteins
an
(high-density lipoproteins ) and the complement systems [104, 105], disintegration of liposome

membrane formed from dioleoyl phosphatidylethanolamine/oleic acid (DOPA/OA) and leakage of

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drug content has been reported through the action of albumin [106]. Moreover, calcium ion induces

aggregation of liposome composed from DOPE/OA [107, 108], some studies reported that
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incorporation of sphingomyelin in AVE (Artificial Viral Envelopes) liposomes can decrease lysis
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occurrence in serum and increase stability [109]. N-acyl-phosphatidyl ethanolamine

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incorporation into liposomes has shown to assist the liposomal biological stability

against plasma components and also increase the circulating time of the vesicles
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[110]. Previously published study reported that fluorinated liposomes more stable than the

conventional one, it also indicated that lipophobic characteristics of the fluorinated intramembrane

core protects the liposomes, probably by minimizing their interactions with the lipophilic serum

components decreasing their adsorption at the surface and hindering their diffusion into the

fluorinated bilayer, furthermore, the fluorinated liposomes stability in serum accrue from the higher
negative dipole potential of fluorinated membranes that affect the binding of negatively charged

lipophilic ions reducing the binding of the serum proteins (negatively charged) that penetrate into

the interfacial region of liposomal bilayer [111], moreover, the fluorinated liposomes showed

longer systemic half-lives in comparison with the conventional DSPC and DSPC/cholesterol

liposomes [112]. The binding of polyethylene glycol on the liposome surface was found to decrease

the liposome clearance and reducing the liposome uptake by the phagocyte system [113, 114].

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Finally, the biological stability of liposome in blood and other biological fluids still needs further

investigation.

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5 Applications of liposomes

5.1 Liposomes for anticancer drug and gene delivery. an

Due to the nature and behavior of cancer tissue, and the large difference between it and the
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normal one, the cancer tissue considered an appropriate target for drug delivery system, for

example, the tumor vasculature is characterized by leaky vasculature and limited lymphatic
d

drainage [115], consequently, macromolecules and micromolecules can be easily accumulated in

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the intercellular spaces of a large variety of tumors. Furthermore, there are specific markers that are

not present in the normal tissue blood vessels (aminopeptidase and N, integrins) [116, 117]. A
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continuous angiogenesis in the cancer tissue acts as a target for anticancer drug vectors; once the
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anticancer drug delivery systems reach the extracellular spaces, the active drug can be released by

different mechanisms, such as the pH difference between the blood (7.4) and the extracellular

spaces of cancer cells (4-5 acidic PH), which can affect the acid sensitive polymers carrying the

anticancer drug. Another mechanism is the exposure of anticancer drugs carrying polymers to

degradation by the lysosomal enzyme [118].

Liposomes are used as a carrier for numerous anticancer drugs in the conventional or modified

forms (PEGylated liposome). In a recent study, folic acid (FA)-conjugated liposome was designed

to co-encapsulate both celastrol (Cs) and irinotecan (Ir) and its effect in breast cancer therapy was

investigated. DPPC and DSPE-PEG-NH2 were used for liposome preparation, the formulation

showed small vesicle size (190 nm) with narrow PDI (0.1) and better drug release profile at pH 5.0

for both drugs, furthermore, it exhibited higher cellular uptake and improved apoptosis in breast

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cancer cells (MCF-7 and MDA-MB-231) [119]. The pharmacokinetic property of doxorubicin was

significantly enhanced and its anticancer effect against human lung carcinoma A549 cells was

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improved upon its encapsulation into selenium coated liposome (Dox-SeLPs), lecithin S100,

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DOTAP, and cholesterol were used for liposomal preparation by ammonium sulfate ((NH4)2SO4)
an
gradient method, Dox-SeLPs displayed higher in-vitro cytotoxicity on A549 cells with significantly

lower IC50 ( 0.92 ± 0.16 μg/mL) than that of free (4.40 ± 0.58 μg/ mL) and liposomal doxorubicin
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(5.68 ± 0.73 μg/mL) [120]. Antibody fragment (AF)-combined liposome was used for

encapsulation of gemcitabine (GEM) and paclitaxel (PTX). phospholipids used for the preparation
e d

of liposome were DPPC, DSPE-PEG2000, and cholesterol, the drug-loaded liposome displayed a
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particle size about 135 nm with a narrow PDI. The cytotoxic effect of the liposomal formulation
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(AF-GPL) in pancreatic cancer cells was evaluated, it showed higher cytotoxicity effect with IC50

value of 0.45 μg/ml significantly lower than that of gemcitabine (5.9 μg/ml), paclitaxel (4.2 μg/ml),
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and GEM/ PTX co-loaded liposome (1.92 μg/ml) [121]. cationic liposomes were used as a drug

carrier for STAT3 siRNA and curcumin for topical treatment of skin cancer [122]. Thin film

hydration technique was used for the preparation of liposomes, in which curcumin was mixed with

1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE), 1,2-Dioleoyl-3-trimethylammonium

propane (DOTAP), and C6 ceramide. After that, methanol was used to dissolve the mixture. A
rotary evaporator was used to evaporate the solvent, and formation of lipid thin film with the drug,

then using 20 mM buffer at pH 7.4 for film hydration. Finally, complexation of the liposome with

STAT3 siRNA was performed through the addition of curcumin-loaded liposomes to siRNA in the

buffer solution (pH 7.4), then the preparation was vortexed for 30 s, and incubated for 20 min at

room temperature. This preparation was used for growth inhibition of B16F10 Melanoma cells. The

data showed a great inhibition of B16F10 cancer cell growth (76.3±4.0%) by using curcumin-

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loaded cationic liposome- STAT3 siRNA complex (250 μM curcumin and 0.5 nM siRNA)

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compared with individual agents (free STAT3 siRNA and free curcumin). Another study reported

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the application of liposomes for improving the penetration of siRNA through the stratum corneum

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and epidermis for melanoma cells treatment by using the solvent dispersion technique for liposomes
an
preparation [123]. DOTAP solution (1,2-dioleoyl-3-trimethylammonium propane chloride

dissolved in chloroform10 mg/ml) was mixed with a NaChol solution (sodium cholate in ethanol 10
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mg/ml). Then, the lipid film was formed after the evaporation of the solvent under vacuum,

followed by hydration of the film in buffer (pH 7.4). The data of this study indicated that the
e d

developed lipoplexes had the ability to penetrate the layers of the skin entering basal epidermis cells
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and inhibit the target proteins expression. Different lipid material (DOPC/SM/Chol/DOPS/DOPE)
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were used for exosome preparation intended for VEGF (vascular endothelial growth factor) siRNA

delivery to human lung carcinoma A549 cells, exosomes exhibited enhanced storage stability and
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anti-serum aggregation effect and also showed significantly higher cellular uptake and silencing

efficacy in comparison with free siRNA and PC-Chol liposomes [124]. The surface modification of

the liposomes such as cationic liposomes was found to be effective Nanocarriers in-vitro, but

unfortunately, the in-vivo study showed cytotoxicity. Therefore, modification of neutral PEGylated

liposomes with cell penetrating peptides (CPPs) was performed to improve the cellular uptake
[125]. In this study, ethanol injection technique was used for liposome preparation, showing

spherical morphology and uni lamellarity of each liposome class with small size vesicles 50-150

nm. The obtained data proved that R8-PLPs (Octaarginine (R8) peptide PEGylated liposomes)

enhanced cellular association, negligible cytotoxicity, and had higher capacity in gene silencing as

compared to the cationic liposomes that showed significant cytotoxicity. Various liposome

formulations for gene delivery and diverse types of anticancer drugs are shown in Table 2.

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5.2 Applications of liposomes in vaccine formulation

Liposomes are flexible drug delivery systems that can be surface-modified with various

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molecules. The encapsulation of peptide antigens or viral membrane proteins into the liposomes

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has been shown to produce humoral and cell-mediated immune response, and generate solid and
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durable immunity against the pathogen [126, 127]. For example, Epaxal® vaccine for hepatitis A,

which considered a successful commercial product based on the liposomal formulation as a drug
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carrier was approved for human use in Switzerland in 1994 [128]. A Recent study explained how

the composition and structure of liposomes demonstrate the impact of the MPER peptides
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(membrane-proximal external region A promising HIV antigen) on the strength and durability of
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the humoral responses and T-helper cell responses in mice [129]. The MPER of HIV-1 gp41 is a
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peptide expressed on the virion surface. It is considered as the most important vaccine recognized

and neutralized by many broadly neutralizing antibody [130]. But this antigen has poor
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immunogenicity and need to be presented on membranes to physiologically matching the HIV

native virus and to enhance the immune response. Liposomes were formulated by mixing [

DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine): DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1′-

rac-glycero1) sodium salt)], palm-MPER and DSPE-PEG in chloroform, then the organic solvent

was evaporated under vacuum, followed by the hydration of the lipid film by using phosphate
buffer. The obtained liposomes undergo vortexing then freeze-thawing cycles, followed by

polycarbonate membranes extrusion. The antigen immunogenicity was affected by various factors,

for instance, immunoglobulin G (IgG) titers specific to MPER is decreased by 15-fold and 20-fold

upon reduction of liposomes particle size from 200 nm and 150 nm, respectively to the particle size

of 65 nm. Also, the immunogenicity is oppositely proportional to the liposome membrane fluidity,

and this confirmed through mice immunization with liposomes with lower membrane fluidity

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(DMPC (Dimyristoylphosphatidylcoline), DOPC and DOPG) and highly fluid membrane (DOPC

and DOPG) demonstrated that a higher IgG titer about 6.2-fold obtained through immunization by

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the DMPC-containing liposome. Furthermore, it was observed that the immunization was enhanced

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by increasing the density of the antigen on the surface of the liposome while increasing the antigen
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density to 2000 MPER / liposome led to fewer antibody titers [131].

Another study proved that DPC (dimethyl dioctadecyl ammonium, Poly I: C and
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cholesterol) can be used as adjuvant forming a unique type of cationic liposome for delivery of
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Tuberculosis specific antigen protein. The study revealed that the formulation stability was affected
e

upon incorporation of the oppositely charged Poly I: C into liposomes dioctadecyl ammonium
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(DDA), and consequently limitation of the clinical application. So, for stability improvement,
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cholesterol and gelatin incorporation has been investigated. It was observed that the in-vitro

stability increased along with the addition of gelatin and cholesterol, but unfortunately, the addition
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of gelatin to the AMM TB (subunit vaccine Ag85B-MPT64 (190-198)-Mtb8.4) antigen in adjuvant

to DP resulted in decreasing the protective efficacy of the AMM vaccine. In contrast, cholesterol

addition into DP did not decrease the protective efficacy or the immunogenicity of the vaccine.

Furthermore, the cholesterol addition into the membrane of the liposomes improved the packing of

the lipid, and consequently decreased the phase transition temperature, resulting in in-vivo and in-
vitro stability improvement of the liposomal formulation. Finally, the novel stable adjuvant DPC

had the ability to sensitize the immune response of Th1-type cell [132]. Table 3 shows a liposomal

formulation of different substances used for the generation and potentiation of the immune

response.

5.3 Applications of liposomes in ophthalmology

Drugs applied topically in the form of ointment, solutions, and suspensions are used for the

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management of most ophthalmic disorders. As a result of various pathophysiological and

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anatomical barriers found in the eye, these dosage forms have poor ocular bioavailability. For the

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management of eye syndromes including the posterior and anterior segments, liposomes were used.

For example, dry eyes have been treated through liposomal aqueous suspension [133], also novel
an
spray has been prepared in which phospholipid liposomes are delivered to the tear film [134].

Liposomal formulation for keratitis has been developed, for example, eye drops of amphotericin B
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(AmBisome®) would be proper and convenient for fungal infections
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with Curvularia, Fusarium, Candida, and Aspergillus that can cause corneal serious ulceration
e

[135]. Another formulation of liposomes for corneal transplant rejection, endophthalmitis, uveitis,
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and proliferative vitreoretinopathy has been investigated.

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Ciprofloxacin (Ciprocin) is a commercially available eye drops, active against both aerobic

gram-positive and gram-negative bacteria. By making a comparison between ciprofloxacin eye

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drops and ciprofloxacin liposomal formulation using the Albino rabbit as an animal model, it was

observed that the maximum aqueous humor concentration (Cmax) was 3.87 mcg/mL from the

liposomal formulation compared with 2.68 mcg/mL for ciprocin eye drops nearly with the same

Tmax. Furthermore, the values of liposome formulations (area under the curve) were significantly

higher than that of ciprocin eye drops, and this refers to the higher ocular absorption, and
consequently bioavailability for ciprofloxacin delivered by liposome in comparison to the eye

drops. As a result, the encapsulation of ciprofloxacin into liposomal formulation leads to higher

bioavailability, residence time enhancement, and reduction in the dose of the drug with a higher

chance for penetration through the tissue of the eye [136]. A novel study made an intercalation

between betaxolol hydrochloride (BH) (an effective drug for glaucoma) and Montmorillonite (Mt)

(ion-exchange drug carrier with the large surface area and perfect exchangeability) and then

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incorporated into liposomes (Mt-BH-LPs) as an ocular drug-delivery system. A slower release was

observed from Mt-BH-LPs (50.3%) compared with the betaxolol hydrochloride sample that showed

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approximately 100% rapid release within 2.5 h. Also, by using the Draize method for the evaluation

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of the irritation effects of the formulations on the rabbit eye, no irritation effects attributed to Mt-
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BH-LPs was observed. Furthermore, the in-vitro pre-corneal retention test demonstrated that the

highest BH concentration (38.87μg/mL) was immediately observed and rapidly decreased to

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undetectable values within 60 min after application of BH sample, while the concentration of

betaxolol reached undetectable values at more than 110 min after the application of Mt-BH-LPs.
e d

Also, the in-vivo test confirmed these in-vitro results, the tears BH concentrations from Mt-BH-LPs
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were three-fold larger than those of BH solution within 30 min. Moreover, the concentration of
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betaxolol (13.6μg/mL) in Mt-BH-LPs was detected at 240 min. These results together revealed that

Mt-BH-LPs could be able to prolong the drug retention time on the eye surface, and also decreased
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drug elimination [137].

5.4 Liposomes in wound healing

Liposomes were proved to be helpful in enhancing the stability, local deposition and

permeation into the skin and deeper tissues and extending the release of their contents. Furthermore,
it can offer a moist environment on the surface of wound skin because of liposome effective closure

as epidermic cells, which lead to wound healing [138].

There are many substances that cannot penetrate through the skin as a result of their

physicochemical properties, such as hydrophilicity and large molecular weight, which could lead to

lower bioavailability and lower biological response. Using liposomes as drug delivery for these

substances would overcome these problems. External application of growth factors [epidermal

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growth factor (EGF), fibroblast growth factor (FGFs), vascular endothelial growth factor (VEGF),

and platelet-derived growth factor (PDGF)] has good impacts on wound healing. One of these

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growth factors is the basic fibroblast growth factor (bFGF) has the ability to stimulate

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differentiation and proliferation of fibroblast, and also promote angiogenesis formation, which
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consequently improves wound healing [139]. Lower stability, short half-life (bFGF about 1.5 min),

and easy degradability (enzymatic degradation) of these growth factors could cause insufficient
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efficacy. Liposomes have been used to encapsulate bFGF to improve its stability; however, it was

exhibited to rapid leakage when it was topically applied due to the liquid status of liposomes. So,
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preparation of liposome with the hydrogel core of silk fibroin including the bFGF growth factor has
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been developed to prevent the rapid leakage of the bFGF. By this formulation, bFGF stability in the
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wound exudate was maintained, penetration ability was enhanced, and consequently, its cell

proliferation activity and wound healing ability were promoted [140].

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Cysteine protease is an enzyme derived from Carica papaya fruit with higher proteolytic

activity; it promotes the skin exfoliation and produces soft skin through fibrosis removing. But its

large molecular weight and hydrophilic nature limit its use for the topical application. Liposomes

were used for the encapsulation of papain enzyme in order to overcome these drawbacks. By

making a comparison between different papain formulations (papain solution, papain- propylene
glycol physical mixture, and conventional liposomes) and propylene glycol liposomal formulation,

the results approved that the propylene glycol liposomes possessed the higher ability for the

reduction of fibrosis (5.566, 3.509, 2.242 times, respectively). This indicated that the propylene

glycol liposomes increased the deep permeation ability of the enzyme into the skin layers of second

degree burned rats [141]. Various substances encapsulated into liposomes for their application in

wound healing are shown in Table 4.

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5.5 Application of liposomes in miscellaneous dermatological disorders

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Acne vulgaris is a prevalent dermatologic disease with substantial cutaneous and

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psychological disease burden; it is mostly dispersed in adolescents. The pathogenesis of acne

mainly depends on four factors, stimulation

an of pilosebaceous gland by androgen

(dihydrotestosterone), hyperkeratinization of the follicle, the skin microbial flora colonization

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(Propionibacterium acnes), and inflammation [142].

Due to the severe systemic side effects of the tretinoin (TRT) [143], it is mostly applied as a
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topical formulation. However, the effectiveness of TRT as a topical preparation (creams, lotions,
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gels, and emulsion) is limited because it had higher sensitivity to light and air, and consequently

limited stability and loss during storage. Also, the barrier properties of the skin hinder the
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penetration and the deposition of the drug into the skin layers. Accordingly, several studies have
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been carried out to incorporate TRT into liposomes to overcome its problems. Liposomes were

prepared by an ether injection technique since dichloromethane was used to dissolve the lipid

components and TRT, then the solution was injected slowly into distilled water (100 mL) under

stirring condition. Liposomes were also prepared by thin film hydration method, the lipid

component, the drug and cholesterol were dissolved in dichloromethane, and then rotary evaporator

was used to evaporate the solvent followed by hydration the lipid film with distilled water. The
study reported that insignificant erythema (0.2±0.37) was observed for liposomal gel formulation.

In contrast, TRT gel (0.025%) showed significant erythema (1.70±0.751). The marketed product

caused an erythema score of 1.40±0.534 and was not able to reduce the irritation resulted from the

topical application of TRT. The liposomal formulation possessed a zeta potential of - 41.2±1.2mV,

this large value refers to the highest stability of the prepared liposomes since the aggregation of

liposomes was prevented by the strong electrostatic repulsion interaction [144].

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P. acnes produce extracellular lipase enzyme, a novel study prepared liposome loaded with

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lipase-sensitive singlet oxygen-producible (pullulan-pheophorbide a (PU-Pheo A)) and

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erythromycin (LSSPL). Firstly, the erythromycin liposomes were prepared by the thin film

hydration technique, and then the pullulan-pheophorbide A conjugates (1 mg/mL of Pheo A) was
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added to the erythromycin liposomes and stirred overnight. The study showed that the lipase

enzyme produced by the P. acnes induced structural breakdown of LSSPL and liberate of the
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encapsulated erythromycin. It also reported that the using of irradiation by laser onto free Pheo
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improved the LSSPLs antibacterial activity. The in-vitro study showed that LSSPLs had greater anti
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bactericidal properties and reduced viability [3.12 log of CFU (99.92%)] compared to OELLs (only
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liposome containing erythromycin) [1.99 log of CFU (98.98%)] and OPCLs (liposome coated with
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Pheo A) [1.58 log of CFU (97.40%)] under laser irradiation conditions. Reduction in inflammation

caused by the P. acnes in mice skin showed that the OELL application leads to an average skin
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volume decrease to 31.4%, OPCLs treatment using laser irradiation reduced the volume of the skin

by 37.3%, but the LSSPL application with laser irradiation showed superior reduction in the

inflamed skin to 5.5% after 7 days [145].

Psoralen combined with ultraviolet A radiation (PUVA) was recommended by FDA for the

treatment of severe psoriasis. But the lower permeability and poor deposition of psoralen into the
skin layers, and the side effects related to conventional psoralen dosage forms (severe burning,

pigmentation, and blisters) decreased the therapeutic efficiency and safety of topical PUVA.

Anionic and cationic liposome-encapsulated psoralen by thin film hydration technique was

prepared. The ex-vivo test confirmed that cumulative skin permeation of psoralen was improved

about 5-fold difference with liposomal formulation gel [cationic liposome (9.18±0.07%], anionic

liposome [9.28±0.65%)] in comparison to the solution form of psoralen (1.90±0.18%) in 24 h. An

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in-vivo study in a murine model with psoriasis induced by Imiquimod showed that the mouse

groups treated with liposomal formulation gel showed the highest improvement of psoriasis

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symptoms (thickening, erythema, and scales) in comparison to PSR-Solution due to greater skin

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permeation of psoralen from liposomal carriers [146].
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Another study made co-encapsulation of resveratrol and psoralen for evaluation of anti-

oxidant and PUVA combination in the management of vitiligo. In this study, mouse melanoma cells
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(B16F10) was used to evaluate the effect of the prepared formulation due to the low incidence of

vitiligo disease that may cause time-consuming, and the high cost of vitiligo model preparation. The
e d

study observed that co-encapsulation of the two drugs into the ultra-deformable liposomes resulted
pt

in higher stimulation of melanin and tyrosinase activity by psoralen and significant anti-oxidant
ce

activity by resveratrol [147].

6 Present prospective and future challenges

Ac

In the pharmaceutical application, using of liposomes as a drug delivery system for specific

targeting, sustained, and/or controlled release, as well as for immunization, was and still is the

driving force for innovative technologies development. Surface modification of liposomes provides

many targeting approaches and ligand density optimization on liposomes surface can increase the

liposomal uptake in tumor cells. However, increasing the density and the length of the ligand
beyond optimum conditions will lead to aggregation and steric hindrance that will influence ligand-

receptor affinity and interaction. So, the optimum density and length of the ligands and their impact

on the liposomal size, the circulation of liposomes in the blood and the affinity of the ligand to the

targeted receptors should be carefully addressed [148, 149, 150]. Stability of the liposomal

formulation and the leakage of the active components from vesicles after administration are vital

problems which need to be monitored to get the optimum therapeutic effect, especially in tumor

t
ip
therapy. phospholipids used in liposome preparation are very susceptible to oxidation and

hydrolysis, so it has a short shelf life. And also, the electrostatic stabilization of the vesicles cannot

cr
offer acceptable stability to liposomes used for encapsulation of substances highly susceptible to

us
disintegration such as enzymes and proteins intended for in-vivo applications. The liposome
an
solutions sterility is another great challenge that should be considered, sterilization is typically

carried out using filter membranes to avoid degradation caused by other techniques, for instance, γ-
M
irradiation, ultraviolet (UV) and dry heat sterilization, unfortunately, filtration techniques

characterized by more time consuming and lower efficiency for removal of viruses [151], moreover,
e d

because of the higher viscosity of the liposomal preparations that can lead to premature membrane
pt

blocking and also, the lower surface tension of these solutions affects the contact angle with filter
ce

membrane and decreases bubble point increasing the probability of bacterial penetration through

these membranes [152]. Several preparation techniques were developed, however, most of them are
Ac

suitable for small-scale and laboratory application and less for the large-scale production.

Unfortunately, the availability of certain production techniques as well as the quality issues depends

on the properties of the lipids themselves. This limits the choice of liposome kinds from which one

can select when improving liposome-based drug therapy. Severe control of the product quality is

essential to achieve the predictable therapeutic effect, quality control related to unwanted by-
products, such as organic solvents residues, degradation products, lysolipids, and other lipid

detergents are just as vital as pyrogen-free and sterile conditions.

Development of liposome as a drug delivery system to control drug release rates and

enhance disease-specific targeting has shown potential impact in global healthcare. However, there

has been a general deficiency of precise protocols to characterize the liposomal drug product at

physicochemical, physiological and biological levels. The following aspects should be considered

t
ip
to support the development of the liposomal product;

cr
- physicochemical properties of a liposomal product are very important to confirm its quality

us
that usually affected by the purity and stability characteristics of phospholipid and other

critical excipients, the active substance/lipid moiety ratio at appropriate manufacturing steps
an
should be within acceptable range to ensure reliable preparation performance, physical
M
morphology, mean vesicle size and polydispersity index, stability of the active ingredient,

lipid materials and other excipients in the final product, including estimation of degradation
d

products (e.g. lysolipid, hydrolytic/oxidated moieties), in-vitro release rate of the active
e
pt

substance from the vesicles in physiologically/clinically related media [153]. also,

identification and control of main intermediates in the manufacturing process have a critical
ce

impact on the product quality.

Ac

- the non-clinical studies to be carried out prior to clinical studies should include reasonable

study of pharmacokinetics (biodistribution, metabolism, and clearance), pharmacodynamics

and toxicology, during the preclinical evaluation, biocompatibility and immunotoxicity

should be taken into consideration along with an appropriate assessment of product

properties during the development includes administration route, dosage regimen, targeted

disease environment, and therapeutic index.

 - development and validation of sensitive analytical methods to quantify drug, metabolite and

other byproducts in blood/tissue will be necessary. The aforementioned criteria along with

the regulatory perspectives of nanomedicine for product development that presented

underlying numerous procedures by the European Medicines Agency (EMA) and Food and

Drug Administration (FDA) can be taken in consideration to support the development and

the marketing approval of liposomal product [154, 155].

t
ip
7 Conclusions

Great attention has been given to liposomes as drug carriers to improve and enhance the

cr
therapeutic index of drugs. Liposomes research has expanded during the last three decades and due

us
to their unique characteristics (non-toxic, biodegradable, biocompatible, non-immunogenic, lowers
an
systemic toxicity, sensitive drug molecules protection, targeted delivery, and improved

pharmacokinetic properties). Liposomes have a wide range of functions and applications including
M
the using of more precise cell ligand-targeted on their surface due to the facile change of structure.

The main challenge is that it is impossible to cross most regular pellicle barriers due to their
e d

imposed size. However, with the advance of liposomes technology, they will play a more important
pt

functional role in the clinical environment. Improving the future researches on the existing
ce

platforms and to address the current translational and regulatory limitations would be achieved by

understanding the advances and developments in liposomes technology, and the overcoming their
Ac

formulation challenges.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81503007

and 21574059).

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Graphical abstract

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The figure showing different application of liposome as a drug delivery system for example in skin disorders
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(skin cancer, burns and infection), ocular disorders (conjunctivitis and glaucoma), cancer therapy (ovarian
cancer), and vaccine delivery (tuberculosis).
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 Table1. liposomal formulation recently used in the global liposomes drug delivery market and some others still under

clinical trials.

Active drug Brand name Route of Lipid composition Indication Company

administration
Bupivacaine Exparel® Infiltration, Cholesterol, Interscalene brachial plexus Pacira
interscalene DPPG, and DEPC nerve block to produce post- Pharmaceuticals,
Branchial surgical regional analgesia Inc.
Plexus Nerve following shoulder surgery
Block in adults
Zoster Vaccine SHINGRIX® Intramuscular DOPC and Prevention of herpes zoster GlaxoSmithKline
Recombinant, cholesterol (shingles) in adults aged 50 Biologicals

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ip
Adjuvanted years and older
Irinotecan Onivyde® Intravenous DSPC and Pancreatic Cancer Merrimack
MPEG-2000- Pharmaceuticals,

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DSPE Inc.
Doxorubicin DOXIL® Intravenous Cholesterol, Treatment of ovarian Janssen Products,
hydrochloride HSPC and cancer, AIDS-related L.P.

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MPEG-DSPE. Kaposi's sarcoma, and
multiple myeloma
Vincristine sulfate Marqibo® Intravenous Sphingomyelin
an Acute lymphoblastic Spectrum
and cholesterol leukemia Pharmaceuticals,
Inc.
Cytarabine and Vyxeos® Intravenous DSPC, DSPG, Acute myeloid leukemia Jazz
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daunorubicin and cholesterol Pharmaceuticals plc
Cytarabine DepoCyt® Intrathecal Cholesterol, Intrathecal treatment of Pacira
administration DOPC, and lymphomatous meningitis Pharmaceuticals,
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DPPG. Inc.
Marqibo®, Phase 1 Intravenous Sphingomyelin Relapsed acute Spectrum
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dexamethasone, and cholesterol lymphoblastic leukemia Pharmaceuticals, Inc

mitoxantrone, and (ALL)
pt

asparaginase (UK
ALL R3)
Pomalidomide in Phase 1 Intravenous Cholesterol, Pomalidomide in National Cancer
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combination with HSPC and combination with Institute (NCI)

doxorubicin MPEG-DSPE. doxorubicin liposome in
liposome people with advanced
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refractory Kaposi sarcoma

Daunorubicin- Phase 1/2 Intravenous Cholesterol, Study of CPX-351 alone Children's Oncology
cytarabine (CPX- DSPC, and DSPG followed by fludarabine, Group and National
351) cytarabine, and filgrastim Cancer Institute
for children with relapsed (NCI)
acute myeloid leukemia
(AML)
Cisplatin Phase 1/2 Intravenous MPEG-DSPE and Evaluate the safety and Oncology Venture
egg PC tolerability of LiPlaCis
(liposomal cisplatin
formulation) in patients with
advanced or refractory
 tumors
Cytarabine, Phase 2 Intravenous Cholesterol, Study of the impact of National Cancer
idarubicin, DSPC, and DSPG clinicogenetic risk-stratified Institute (NCI),
decitabine, and management on outcomes University of
liposome- of acute myeloid leukemia Nebraska
encapsulated in older patients
daunorubicin-
cytarabine
farletuzumab Phase 2 Intravenous Cholesterol, Assess the efficacy and Sponsor:
(MORAb 003) in HSPC and safety of farletuzumab Morphotek
combination with MPEG-DSPE. (MORAb 003) in Collaborator:
carboplatin plus combination with Eisai Co., Ltd
paclitaxel or carboplatin plus paclitaxel

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carboplatin plus or carboplatin plus

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pegylated liposomal pegylated liposomal
doxorubicin (PLD) doxorubicin (PLD) in

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subjects with low CA125
platinum-sensitive ovarian
cancer

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Vincristine Sulfate Phase 2 Intravenous Sphingomyelin Relapsed or refractory acute Wake Forest
and cholesterol myeloid leukemia (AML) University Health
an Sciences
pegylated liposomal Phase 2 Intravenous Cholesterol, Combination therapy for M.D. Anderson
doxorubicin, HSPC and patients with localized Cancer Center
bevacizumab, and MPEG-DSPE. triple-negative breast cancer
M
everolimus (TNBC) with tumors
predicted insensitive to
standard neoadjuvant
chemotherapy
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Cytarabine Phase 2 Intrathecal Cholesterol, Cytarabine liposome for University of

DOPC, and adults with acute California, San
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DPPG lymphoblastic leukemia or Diego

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acute lymphoblastic
lymphoma
doxorubicin Phase 2 Intravenous Cholesterol, Treating patients National Cancer
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hydrochloride, MPEG-DSPE, with stage II-III breast Institute (NCI),

epirubicin HSPC cancer that does not have Rutgers Cancer
hydrochloride, estrogen receptors, Institute of New
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carboplatin, and progesterone receptors, or Jersey

paclitaxel large amounts of
(HER2)/neu protein (triple
negative).
Pegylated liposomal Phase 2/3 Intravenous Cholesterol, treating patients with National Cancer
doxorubicin HSPC and recurrent ovarian, fallopian Institute (NCI)
hydrochloride with MPEG-DSPE. tube, or primary peritoneal
atezolizumab and/or cancer
bevacizumab
Irinotecan Phase 2/3 Intravenous DSPC and Study of Irinotecan injection Ipsen Medical
MPEG-2000- (ONIVYDE®) versus Director
DSPE Topotecan in Patients with
Small Cell Lung Cancer
 Doxorubicin Phase 3 Intravenous DPPC, MSPC and Study of ThermoDox® in Celsion Corporation
(ThermoDox®) DSPE-PEG2000 hepatocellular carcinoma (NASDAQ:CLSN)
(HCC) Using standardized
radiofrequency ablation
(RFA)
Vincristine Phase 3 Intravenous Sphingomyelin Improvement of outcome Spectrum
liposome, Rituximab and cholesterol and reduction of toxicity in Pharmaceuticals, Inc
elderly patients with CD20+ University Hospital,
aggressive B-cell Saarland
lymphoma

DPPG, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1-racglycerol) (sodium salt); DEPC, 1,2-dierucoyl phosphatidylcholine;

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DOPC, dioleoyl phosphatidylcholine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; MPEG-2000-DSPE,

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Cholesterol N-(carbonyl-methoxy polyethylene glycol-2000)-1, 2-distearoly-sn-glycero-3-
phosphoethanolamine; HSPC, hydrogenated soy phosphatidylcholine; DSPG, distearoyl

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phosphatidylglycerol; egg PC, egg yolk phosphatidylcholine; HER2, human epidermal growth factor
receptor 2.

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 Table 2. Liposome for gene and anticancer drugs delivery

Anticancer Lipid component/ Method of

Application Reference
drugs/gene polymers preparation
siRNA DOPC, DSPE-PEG, Ethanol injection Enhancing cellular association and gene [1]
STR-R8 silencing capacity exhibited by R8-PLPs
Anti-VEGF DSPC, PEG-DSPE, Thin layer Cellular delivery of anti-VEGF siRNA in [2]
siRNA DOPE hydration method SKBR-3 breast tumor cell line
(bevasiranib)
siRNA PEG-DSPE, Thin-film Enhancing cellular uptake of siRNA and gene [3]
DOTAP, NIPAAm, hydration method silencing activity by using temperature-
DMAPAAm responsive liposome
Paclitaxel and LHSSG2C14, Film dispersion Overcoming PTX resistance and improving the [4]
anti-survivin LHG2C14 method antitumor effect of PTX and down-regulation of

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siRNA survivin overexpression

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Anti-miR-191 Lipoid S 100, stearyl Thin-film Efficient delivery of anti-miR-191 in SA [5]
amine (SA) hydration method liposome complex for inhibition of breast cancer
cells

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Plasmid DNA EPC, DOPE, Thin film hydration Formation of pDNA/protein /liposome [6]
(pVAX1GFP) DOTAP method complexes for more efficient pDNA delivery

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pDNA and DOPE, DPPC, Poly Thin film hydration Using PEI based, liposome for Improvement [7]
siRNA ethylenimine method transfection efficiency and low cytotoxicity
siRNA DPPC, DPPG, Thin film hydration Reverse doxorubicin resistance of [8]
(silencing DPTAP, PDADMAC method OVCAR8/ADR cells (ovarian cancer).
an
ABCB1 gene),
Doxorubicin
Antimycin A DOPE, cholesterol Thin film Enhancement of the effective delivery and [9]
dehydration and stability of the incorporated drug to the
M
rehydration method mitochondria of A549 cells.
pDNA DOPE, OH-Chol Thin film hydration Explaining the size-dependent endocytosis [10]
method, pathway and the intracellular trafficking of
ultrasonication cationic lipoplexes using bone marrow-derived
d

methods dendritic cells (BMDCs).

Poly(L-lysine)- PC: DSPE-PEG, Thin film hydration Increasing gene silencing efficiency and [11]
e

siRNA complex EpCAM antibody method targeting specificity in breast cancer.

PAC1Rantisense DOPC, DOTAP, Thin film hydration Using light-responsive delivery strategy to [12]
pt

oligonucleotides Verteporfin method achieve enhanced endosome and lysosome

escape and PAC1Rgene silencing in PC12 cells
HIF-1α siRNA Soybean lecithin Thin film hydration siRNA-fc-LPs reduce the production of HIF-1α- [13]
ce

(S100), Folic acid, method associated protein and induce the apoptosis of
oleic acid, hypoxia-tolerant melanoma cells.
cholesterol, chitosan
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Anti-MUC4 DPPC, DSPE, PEG Thin film hydration Higher targeting affinity and improving [14]
antibodies, method antiproliferative effect in PDAC
Gemcitabine
Gemcitabine DOPE, EPC Freezing/thawing Significant enhancement of the cytotoxicity, [15]
method anti-migration, and anti-colony formation
abilities of GEM through targeting of CD44
expressed on BCSCs by HA-conjugated
liposomes
Methotrexate PC, PI Thin film MTX-DG liposomes showing higher plasma [16]
diglyceride ester hydration, concentration, lower toxicity and retarded
freezing/thawing lymphoma growth rate as compared with MTX
method in mice bearing T-cell leukemic lymphoma.
Methotrexate PC, CH, PEG2000‐ Reverse‐phase Reduction the proliferation of human [17]
PE evaporation, thin lymphoblastic cell line K562 and significant
 film hydration inhibition of RNA synthesis
method
Carfilzomib and Methoxy PEG2000- Thin film hydration Explaining Synergistic effect dual drug loaded [18]
Doxorubicin DSPE, DSPC, method liposomes in vitro and their efficacy in inhibiting
DPPE- glutaryl tumor growth in vivo reducing systemic toxicity
on multiple myeloma tumor cells.
Pemetrexed DOPC, DOPE, Reverse-phase Evaluation of liposomal membrane fluidity and [19]
HSPC, POPC, PEG evaporation its effect on drug release inside the murine
mesothelioma-xenograft model
Doxorubicin DSPC, DSPE-PEG Ethanol injection Stealth PoP liposomes provide long circulation [20]
method half-life and higher stability in storage for
months for the anticancer drug
Doxorubicin EPC, DOPE, PEG, Rotary evaporation Dual-targeted liposomes represent an effective [21]
Rhodamine folic followed by freeze cytotoxic formulation and higher tumor growth

t
acid, transferrin drying inhibition in human cervical carcinoma and

ip
A2780-ADR ovarian carcinoma cell monolayers

cr
siRNA, short interfering RNA; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DSPE-PEG,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000];

us
STR-R8, stearylated octaarginine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DOPE,
1,2-dioleoyl-sn-glycero-3- phosphoethanolamine; NIPAAm , 2.1.1 N-isopropylacrylamide;
DMAPAAm, N,N’-dimethylami- nopropylacrylamide; DOTAP, N-[1-(2,3-

double-stranded RNA virus;

an
dioleoyloxy)propyl]-N,N, N-trimethylammonium methyl-sulfate; Reovirus, a non-enveloped
LHSSG2C14, ditetradecyl 2- (4-(2-(2-(2-(2-(2,6-
diaminohexanamido)-3-(1H-imidazol-4-yl) propanamido) ethyl) disulfanyl) ethyl amino)-4-
M
oxobutanamido) pentanedioate; LHG2C14, ditetradecyl 2-(2-(2,6-diaminohexanamido)-3-(1H-
imidazol-4-yl) propan-amido) pentanedioate; Anti-miR-191(microRNA); DPPC,
dipalmitoylphosphatidylcholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1-racglycerol)
(sodium salt); DPTAP, 1,2-stearoyl-3-trimethylammonium-propane(chloride salt);
d

PDADMAC, poly(diallyldimethylammonium chloride); OH-Chol, cholesteryl-3β-

e

carboxyamido ethylene-N-hydroxyethylamine; EpCAM, epithelial cell adhesion molecule;

HIF-1α, hypoxia-inducible factor-1α; siRNA, small interfering RNA; PDAC, pancreatic
pt

ductal adenocarcinoma; HSPC, hydrogenated soy phosphatidylcholine; POPC, palmitoyloleoyl

phosphatidylcholine; BCSCs, breast cancer stem cells; PI, phosphatidylinositol; LPC,
lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol.
ce
Ac
Table 3. Different substances incorporated into liposomes used for generation and potentiation of the immune
response

API Material Disease/purpose Route of Application References

used in the administration
liposome
composition
H5N1 vaccine JVRS-100 H5N1 Intramuscular Addition of JVRS-100 to H5N1 vaccine- [22]
(pDNA with enhanced immunogenicity and cross-
DOTIM/ protection against lethal H5N1 virus disease
cholesterol) in ferrets
SLP and DOTAP and Antigen delivery Intradermal Liposome enhances the delivery of the SLP [23]
poly(I:C) DOPC inducing an and to DCs in vitro and induced a functional

t
immune subcutaneous CD8+ T cell immune response in vivo to the

ip
response immunization CTL epitope present in the SLP.
P5 peptide and DOTAP and Murine tumor Subcutaneous Formulation enhance the release of [24]
Poly (I: C). cholesterol Interferon (IFN)-γ and induce strong

cr
antitumor responses
SP-Ld with FA Lecithin and Visceral Subcutaneous Liposomal formulation induces a long- [25]
cholesterol leishmaniasis lasting protective immune response in mice

us
and in hamsters against a challenge from
virulent L. donovani.
(IRV) HSPC and Rabies Intraperitoneal Liposomal rabies vaccine showed higher [26]
cholesterol injection
an survival rate and enhancement of the
immune response of mice to rabies vaccine
(Higher levels of
interleukin-2 (p < 0.05), interferon-c (p <
0.01), and natural killer cell activity (p <
M
0.05))
Lipopeptide- DDAB, Group A Intranasal Lipopeptides entrapped by liposomes induce [27]
based vaccine DPPC Streptococcus both mucosal and systemic immunity (higher
d

candidates IgG and IgA antibody titres) and also

against GAS produce more balanced and long lasting
e

Th1/Th2 responses in mice

Class B CpG DOTAP: Infectious Intranasal Cationic liposome harboring CpG ODN [28]
pt

ODNs DC- diseases induces both antigen-specific mucosal IgA

cholesterol responses and balanced Th1/Th2 responses.
Anti-Nicotine DOTAP, Nicotine Subcutaneous NsL NPs based anti-nicotine vaccine induce [29]
ce

Vaccine ( DSPE- PEG dependence high titer and high affinity of nicotine-
O-succinyl- 2000 specific antibodies in mice providing a
3hydroxmethyl- promising candidate in treating nicotine
(±) nicotine ) dependence
Ac

CRX-601 DOPC, PE- Influenza Sublingual The coating of modified liposomes produced [30]
PEG2K the most effective specific sublingual
immune response against influenza
Hepatitis B DPPC, Hepatitis B virus Transcutaneous (DMA)-based TCI system loaded with [31]
DNA vaccine cholesterol, immunization cationic liposomes help in improving the
DDA via immunogenicity of DNA vaccine.
microneedles
F1-V DOTAP, Plague Intranasal Liposome-polymer hybrid NPs stimulates [32]
DOPE, H A, stronger humoral and cellular immune
PEG responses. And provide Intranasal
vaccination against Yersinia pestis
GPC3-derived DOPE, Hepatocellular Intradermal Vaccination with pGPC3-liposome inhibited [33]
CTL epitope DOPG, carcinoma GPC3-expressing tumor growth in
peptide DOPC, hepatocellular carcinoma
cholesterol
AMM DDA, Poly Mycobacterium Subcutaneous AMM Liposomal formulation has a good [34]
I:C, tuberculosis stability and induces Th1-type cell-mediated
cholesterol immune responses. and also provide long-
(DPC) term protection against mice M. tuberculosis
infection
Gp2 DMPC, Breast cancer Subcutaneous Liposomal formulation showed the highest [35]
DMPG, number of IFN-γ+ in CD8+ cells and
DOPE, cytotoxic T lymphocyte response.
cholesterol The immunization led to lower tumor sizes
and longer survival time against a breast
cancer model overexpressing
HER2/neu.

t
S19-OMP DOPE, Brucella abortus Subcutaneous (S19-OMP-liposome) showed enhanced [36]

ip
DODAP infection protection compared to groups of mice
inoculated with S19 OMP alone and S19 live
B. abortus vaccine with higher immune

cr
response (Th1 based cellular immunity)
PCV-2 Soybean Porcine Subcutaneous RGPL showed excellent particle stability [37]

us
attenuated phospholipid, circovirus type 2 with a strong IgG response and increase the
antigen cholesterol, infection production of Th1 and Th2 associated IgG
RGPL subtypes and cytokines
an
Poly (I:C), polyriboinosinic: polyribocytidylic acid; DOTAP, dioleoyl-3-trimethylammonium propane;
M
SP-Ld, L.donovani intracellular serine protease; FA, Freund’s adjuvant; IRV, inactivated rabies
vaccine; DDAB, dimethyldiocta decyl ammonium bromide; GAS, group A Streptococcus; ODNs,
oligodeoxynucleotides; CpG, containing immunostimulatory motifs; NsL NPs, nanohorn supported
d

liposome nanoparticles; PE-PEG2K, [N-(carbonyl-methoxy polyethyleneglycol-2000)-1,2-distearoyl-

e

sn-glycero-3-phosphoethanol amine sodium salt; DDA, dimethyldioctadecylammonium; DMA,

pt

dissolving microneedle array; TCI, transcutaneous immunization; F1-V, a candidate recombinant

antigen for Yersinia pestis; H A, hyaluronic acid; HCC, hepatocellular carcinoma; GPC3, an oncofetal
ce

antigen overexpressed in HCC; AMM, subunit vaccine Ag85B-MPT64(190-198)-Mtb8.4; DDA,

dioctade-cylammonium; Gp2, a HER2/neu-derived peptide; DMPC, dimyristoylphosphatidylcoline;
DMPG, dimyristoylphosphoglycerol; OMP, outer membrane protein of Brucella abortus S19 vaccine;
Ac

DODAP, dioleoyl-3-dimethylammonium-propane; RGPL, R. glutinosa polysaccharide.

Table 4. Substances encapsulated into liposomes for wound healing

API Liposomal Application References

composition
Danggui Soybean DBLTG formulation was successfully leading to faster wound [38]
Buxue Extract phospholipid and closure, higher hydroxyl proline levels, and provide topical
cholesterol sustained release drug delivery systems.
bFGF Phospholipids, silk SF-bFGF-LIP formulation enhances bFGF stability (More than 50% [39]
fibroin of free bFGF are degraded after 8 h of incubation, while only 18.6%
of the encapsulated bFGF in SF-LIP are destroyed), accelerates the
wound closure of mice with deep second-degree scald.
Gatifloxacin Phospholipon 90 Lyophilized liposomal wafers provided localization and sustained [40]
(GTX) H, cholesterol release of the drug (GTX gel without liposomes 54.53% released
while from the liposomal gel was 33.04% after 12 hrs.) to the

t
wound site.

ip
Hemoglobin PEG, inositol LEH enhances surface blood flow and accelerates skin wound [41]
hexaphosphate, healing in diabetic mice
2,3-

cr
diphosphoglycerate
GHK-Cu DOPC, DOPG GHK-Cu-liposomes enhance the expression of both growth factors [42]

us
(VEGF and FGF-2), and decrease the wound healing time in
comparison with the free GHK-Cu, In a mice scald model
Povidone– Phospholipids Liposomal 3% PVP–I hydrogel provide better scores in subjective [43]
Iodine assessments of quality of wound healing, and can be used as a
an
dressing for non-infected MSGs
Quercetin PC and cholesterol Liposomal formulations produce sustained release of drug in wound [44]
areas (in vitro)
siRNA DOTAP LPP formulation enhances delivery of siRNA targeted against [45]
M
Keap1 and accelerates diabetic tissue regeneration in humanized
Murine diabetic wound.
Cysteine PC, propylene PG-liposomes enhance the deposition of papain and the skin [46]
protease glycol (PG) permeation efficiency in thermal burn-induced wound
d

(papain) Fibrosis.
Madecassoside Egg yolk lecithin, MA double-emulsion liposomes had better stability and more [47]
e

(MA) cholesterol excellent abilities of permeation and distribution in the burned skin
with superior performance in wound healing.
pt

Usnic acid PC, gelatin UAL provide greater collagen. Deposition, development, and [48]
maturation of granulation tissue and scar repair in the porcine
model
ce

DBLTG, Danggui Buxue extract-loaded liposomes in the thermosensitive gel; bFGF, fibroblast
growth factor; LEH, liposome-encapsulated hemoglobin; (GHK)-Cu, glycyl-l-histidyl-l-lysine;
MSGs, meshed skin grafts; LPP, lipoproteoplex.
Ac
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